Compositions and methods for peronospora resistance in spinach

ABSTRACT

The invention provides for spinach plants with broad-spectrum resistance to downy mildew disease and their progeny. Such plants may comprise unique combinations of alleles resulting in the broad-spectrum resistance to downy mildew. In certain aspects, compositions, including distinct polymorphic molecular markers, and methods for producing, using, identifying, selecting, and the like of plants or germplasm with resistance to downy mildew are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/632,871 filed Feb. 26, 2015, which claims the benefit of U.S.Provisional Application No. 61/945,675, filed Feb. 27, 2014, each ofwhich are herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB015US_ST25.txt,” which is 6.2 kilobytes as measured in MicrosoftWindows operating system and was created on Feb. 25, 2015, is filedelectronically herewith and incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of plant breeding and, morespecifically, to methods and compositions for producing spinach plantswith resistance to downy mildew.

BACKGROUND OF THE INVENTION

Downy mildew, caused by the plant pathogen Peronospora farinosa f. sp.Spinaciae (Pfs), is an economically important disease of spinachworldwide, particularly for Spinacia oleracea, the most commonlycultivated spinach species. Currently, fourteen races of the downymildew (DM)-causing pathogen are officially recognized, although newisolates are currently being discovered and named each year. To date, ithas been believed that resistance to DM in spinach was incomplete andrace-specific. The ability of new strains of the pathogen to overcomeresistance in spinach plants therefore makes the development of spinachvarieties with effective levels of resistance to Peronospora farinosa f.sp. spinaciae challenging and increasingly important.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a Spinacia oleracea spinach plantcomprising in its genome allele A, as described herein. In anotheraspect, the invention provides a Spinacia oleracea spinach plantcomprising in its genome a heterozygous combination of alleles thatconfers broad-spectrum resistance to Peronospora farinosa f. sp.Spinaciae. In an embodiment, the broad-spectrum resistance comprisesresistance to at least 10 races of Peronospora farinosa f. sp. spinaciae(Pfs). In another embodiment, the allele that confers broad-spectrumresistance is a combination of alleles which is selected from the groupconsisting of allele A, allele Vt, and allele C. In other embodiments,the combination of alleles comprises alleles A and C and the plant isresistant to at least Peronospora farinosa f. sp. Spinaciae races 7, 8,10, 11, 12, 14, and isolate UA4712; the combination of alleles comprisesalleles A and Vt, and the plant is resistant to at least Peronosporafarinosa f. sp. Spinaciae races 7, 8, 10, 11, 12, 13, and 14; or thecombination of alleles comprises alleles C and Vt, and the plant isresistant to at least Peronospora farinosa f. sp. Spinaciae races 7, 8,10, 11, 12, 13, and isolate UA4712. In another embodiment,representative samples of seed comprising allele A, allele C, and alleleVt have been deposited under ATCC Accession No. PTA-120472, ATCCAccession No. PTA-12486, and ATCC Accession No. PTA-12041, respectively.In further embodiments, the allele A, allele C, and/or allele Vt isgenetically linked to at least one sequence selected from the group SEQID NOs:1-25.

In another embodiment, a plant of the invention comprises an allele A,allele C, and/or allele Vt that shares a genetic source for said allelewith seed deposited under ATCC Accession Nos. PTA-120472, ATCC AccessionNo. PTA-12486, or ATCC Accession No. PTA-12041. In other embodiments, aplant of the invention may be an inbred or a hybrid. In still furtherembodiments, the invention provides a seed that produces such a plant,or a plant part of such a plant. In another embodiment, the plant partis selected from the group consisting of an embryo, meristem, cotyledon,pollen, leaf, anther, root, pistil, flower, cell, and stalk. In a stillfurther embodiment, the invention provides a food product comprising theharvested leaves of such a spinach plant.

In another aspect, the invention provides a Spinacia oleracea spinachplant comprising in its genome a heterozygous combination of allelesthat confers broad-spectrum resistance to Peronospora farinosa f. sp.Spinaciae, wherein one allele confers recessive resistance toPeronospora farinosa f. sp. Spinaciae races 7 and 13. In one embodiment,the broad-spectrum resistance comprises resistance to at least 10 racesof Peronospora farinosa f. sp. spinaciae (Pfs). In other embodiments,one allele is allele Vt and the other allele is allele C, one allele isallele A and the other is allele Vt; or one allele is allele A and theother is allele C.

In another aspect, the invention provides a method of producing aspinach plant with broad spectrum resistance to Peronospora farinosa f.sp. Spinaciae comprising: (a) crossing a first spinach plant comprisingin its genome a first allele selected from the group consisting of A,Vt, and C, with a second spinach plant comprising in its genome a secondallele selected from the group consisting of A, Vt, and C, to produce apopulation of hybrid progeny plants; and (b) selecting at least onehybrid progeny plant from said population that comprises a combinationof said first allele and said second allele that confers broad-spectrumresistance to Peronospora farinosa f. sp. Spinaciae. In one embodiment,the method further comprises production of a population of hybridplants. In other embodiments, the first spinach plant comprises allele Aand the second spinach plant comprises allele C; or the first spinachplant comprises allele A and the second spinach plant comprises alleleVt; or the first spinach plant comprises allele C and the second spinachplant comprises allele Vt. In another embodiment, the invention providesa hybrid progeny plant produced by such a method. In still otherembodiments, the first allele of the plant is allele A and the secondallele is allele C; or the first allele is allele A and the secondallele is allele Vt; or the first allele is allele C and the secondallele is allele Vt.

In another aspect, the invention provides a method of introducingresistance to Peronospora farinosa f. sp. Spinaciae in a spinach plantcomprising: (a) crossing a first spinach plant comprising in its genomea first allele selected from the group consisting of A, Vt, and C, witha second spinach plant comprising in its genome a second, distinctallele; (b) selecting at least one progeny plant that comprises saidfirst allele for resistance to Peronospora farinosa f. sp. Spinaciaebased on the presence in the genome of the plant of a sequence selectedfrom SEQ ID NOs:1-25. In another embodiment, the method comprisesdetecting in the genome of said plant at least two polymorphic nucleicacid sequences selected from the group consisting of SEQ ID NOs:1-25,wherein the presence of the polymorphic nucleic acid sequences areindicative of the presence in the plant of at least two allelesconferring resistance to Peronospora farinosa f. sp. Spinaciae selectedfrom of alleles A, Vt, and C.

In yet another aspect, the invention provides a spinach plant, cell orcell containing plant part comprising at least two polymorphic DNAsequences which are associated with different alleles from among allelesA, Vt, and C. In one embodiment, a spinach plant, cell or cellcontaining plant part are provided comprising at least two DNA sequencesthat are represented in different groups from among those designated(a), (b) and (c), said groups being made up as follows: (a) a DNAsequence comprising SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:22; (b) a DNAsequence comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:12,SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, or SEQ ID NO:22; and (c) a DNAsequence comprising SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, or SEQ ID NO:23. The inventionalso provides a DNA sequence selected from the group consisting of SEQID NOs:1-25.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1—DNA sequence corresponding to scaffold SF63815 anddiagnostic for allele C.

SEQ ID NO:2—DNA sequence corresponding to scaffold SF63815 anddiagnostic for allele A.

SEQ ID NO:3—DNA sequence corresponding to scaffold SF63815 anddiagnostic for allele Vt.

SEQ ID NO:4—DNA sequence corresponding to scaffold SF59002 anddiagnostic for allele C.

SEQ ID NO:5—DNA sequence corresponding to scaffold SF59002 anddiagnostic for allele A.

SEQ ID NO:6—DNA sequence corresponding to scaffold SF59002 anddiagnostic for allele Vt.

SEQ ID NO:7—DNA sequence corresponding to scaffold SF59002 anddiagnostic for allele A.

SEQ ID NO:8—DNA sequence corresponding to scaffold SF59002 anddiagnostic for allele Vt.

SEQ ID NO:9—DNA sequence corresponding to scaffold SF95487 anddiagnostic for allele C.

SEQ ID NO:10—DNA sequence corresponding to scaffold SF95487 anddiagnostic for allele A.

SEQ ID NO:11—DNA sequence corresponding to scaffold SF95487 anddiagnostic for allele Vt.

SEQ ID NO:12—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele C.

SEQ ID NO:13—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele A.

SEQ ID NO:14—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele Vt.

SEQ ID NO:15—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele C.

SEQ ID NO:16—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele A.

SEQ ID NO:17—DNA sequence corresponding to scaffold SF90906 anddiagnostic for allele Vt.

SEQ ID NO:18—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele C.

SEQ ID NO:19—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele A.

SEQ ID NO:20—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele Vt.

SEQ ID NO:21—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele C.

SEQ ID NO:22—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele A.

SEQ ID NO:23—DNA sequence corresponding to scaffold SF34732 anddiagnostic for allele Vt.

SEQ ID NO:24—DNA sequence corresponding to alleles A, C, and Vt ofscaffold SF62749.

SEQ ID NO:25—DNA sequence corresponding to alleles A, C, and Vt ofscaffold SF178637.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows alleles A, Vt, and C, which represent three linkage groupsassembled from genotypes and phenotypes collected for three mappingpopulations, as described in Example 3.

FIG. 2A and FIG. 2B: Show possible breeding methods for development ofhybrids and three-way hybrids.

FIG. 3: Shows sequence alignments of scaffolds containing polymorphismscorresponding to alleles C, A, and Vt. Polymorphisms between alleles areindicated by shading, and can be used to identify and/or diagnose thepresence of downy mildew resistance alleles C, A, and/or Vt in spinach.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for developmentof spinach varieties with resistance to downy mildew (DM). The inventionprovides the identification of three distinct alleles from Spinaciaoleracea, which have been named A, Vt, and C. These alleles can be usedin various combinations to obtain spinach plants with a uniquebroad-spectrum resistance to DM.

The three alleles, designated A from spinach line SMBS011-1162M, C fromSMB-66-1143M, and Vt from SSB-66-1131M, were identified in Spinaciaoleracea and found to provide resistance to existing and emerging DMstrains. Each allele was found to have resistance to a specific set ofraces and/or isolates. The alleles can be used in various combinationsto make hybrids with desired DM resistance.

As shown in further detail herein below, allele C was found to conferresistance to races 1, 2, 3, 4, 5, 6, 7, 8, 10 and newly occurring Pfsisolate UA4712, which is the candidate strain for race 15. Allele A wasfound to confer resistance to Peronospora farinosa (Pfs) races 1, 3, 5,7, 8, 11, 12, 13 and 14. The resistance to races 7 and 13, unlike mostDM races, is inherited in a recessive manner. Allele Vt was found toconfer resistance to races 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 and 13.These newly characterized alleles can be used in novel heterozygouscombinations to obtain spinach plants with broad DM resistance. Thespinach plants may be inbred plants and/or hybrid plants and can includevarious combinations of the alleles as described herein.

For example, in accordance with one embodiment of the invention, allelesC and Vt can be present in a spinach plant (i.e., a heterozygous spinachplant) and provide resistance to Pfs races 1-8, 10-13, and to newlyoccurring Pfs isolate UA4712. In another embodiment, alleles A and Vtcan be present in a spinach plant and provide resistance to Pfs races1-8 and 10-14. Allele Vt complements the recessive resistances fromallele A. Similarly, when alleles A and C are present together in aspinach plant, the plant is resistant to Pfs races 1-8, 10-12, 14 andUA4712. The alleles can be utilized in any combination to providedesired resistance for a particular market or region. For example, ahybrid with alleles C and Vt exhibits resistance to all Pfs races exceptPfs 14. Since Pfs race 14 does not occur in Europe, this hybrid would befully resistant in that geography. As a further example, a hybrid withalleles A and Vt exhibits resistance to all Pfs races except isolateUA4712. New isolate UA4712 is found in limited locations, so this hybridwould be fully resistant in many areas.

In yet another embodiment, a three-way hybrid is contemplated that isresistant to all described DM races and isolate UA4712. In accordancewith the invention, a spinach plant comprising one or two allelesdescribed herein, including, but not limited to, A, C, and Vt, can becrossed with a second, distinct spinach plant comprising a second orthird allele including, but not limited to, A, C, and Vt to produce ahybrid spinach plant comprising a beneficial set of alleles as describedherein.

As further described herein, alleles can be introgressed into selectedspinach varieties in any combination to provide a desired resistance toDM. In accordance with the invention, such alleles conferring resistanceto DM may be introgressed into any desired genomic background of aspecific spinach variety or cultivar. For example, a starting spinachplant containing a given DM resistance allele in accordance with theinvention can be self-fertilized a sufficient number of generations toproduce an inbred spinach variety that is homozygous for the alleleconferring resistance to DM. Such an inbred plant may then be crossedwith another spinach plant that comprises a distinct DM resistanceallele to consistently produce spinach inbreds and/or hybrids thatcomprise a combination of alleles conferring a desirable resistance toDM as described herein. A spinach plant exhibiting resistance to DMaccording to the invention may further be crossed to other spinachplants and selections carried out according to the invention to obtainnew DM-resistant spinach inbreds and hybrids with any desiredcombination of alleles described herein.

Although other alleles conferring resistance to certain races of DM havepreviously been identified in spinach, such alleles have conferredresistance only to a subset of races of Peronospora farinose (Pfs), evenin a hybrid combination. Non-host resistance to DM may also be found inwild relatives of spinach, such as Spinacia turkestanica and Spinaciatetrandra. However, genes from wild relatives often result in negativedrag for commercial characteristics including yield and quality. Theunfavorable alleles found in the wild relatives are often introducedinto the elite germplasm together with DM resistance alleles. Thepresent invention describes novel S. oleracea resistance sources,alleles, and markers that provide race-specific and broad-spectrum DMresistance.

In one embodiment of the invention, alleles A, C, and Vt, conferringresistance to DM may be defined as being on spinach linkage group 6 bycommon markers in sequence scaffolds SF34732 and SF63815 on the distalposition and SFSF59002, SF95487, and SF90906 on the proximal position.Nucleotide sequences associated with and diagnostic for the resistancealleles are provided in SEQ ID NOs:1-25. Polymorphisms between allelesA, C, and Vt are shown in FIG. 3. In other embodiments, allelesproviding broad-spectrum resistance to DM may be defined as from, orsharing, a genetic source selected from accessions SMBS011-1162M,SMB-66-1143M, and SSB-66-1131M, representative deposits of seed of whichwere made with the ATCC under accession numbers PTA-120472, PTA-12486,and PTA-12041, respectively.

The invention further provides methods of producing spinach plants withbroad-spectrum resistance to DM, as well as spinach plants and partsthereof made by such methods. In one embodiment, nucleic acid sequencesor genomic markers may be used to identify alleles according to thepresent invention. These nucleic acid sequences may be used in theidentification of polymorphisms or markers genetically linked in aspinach genome to the DM-resistance conferring alleles in accordancewith the invention. The invention also provides food products derivedfrom such plants and their method of production.

Genetic markers in linkage disequilibrium with DM-resistance alleles ofthe present invention may permit efficient introduction of DM-resistanceinto essentially any spinach genome. This also results in significanteconomization by permitting substitution of costly, time-intensive, andpotentially unreliable phenotypic assays. Further, breeding programs canbe designed to explicitly drive the frequency of specific favorablephenotypes by targeting particular genotypes. Fidelity of theseassociations may be monitored continuously to ensure maintainedpredictive ability and, therefore, informed breeding decisions.

In accordance with the invention, one of skill in the art may identify acandidate germplasm source possessing a desirable DM-resistantphenotype, such as from an accession described herein. One embodiment ofthe invention comprises using the materials and methods of the inventionto obtain an allele conferring broad-spectrum resistance to DM from anyadditional spinach accessions. Using the information set forth herein,including, but not limited to, a sequence scaffold from spinachchromosome 6, DM resistance can be introgressed into any other spinachvarieties.

The development of spinach varieties in a spinach breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines, and the evaluation of the resulting hybridplants. Spinach breeding programs combine the genetic backgrounds fromtwo or more inbred lines or various other germplasm sources intobreeding populations from which new inbred lines are developed byselfing and selection of desired phenotypes. Hybrids also can be used asa source of plant breeding material or as source populations from whichto develop or derive new spinach lines. Plant breeding techniques knownin the art and used in a spinach breeding program include, but are notlimited to, recurrent selection, backcrossing, double haploids, pedigreebreeding, genetic marker enhanced selection, and transformation. Often acombination of these techniques is used. Thus, inbred lines derived fromhybrids can be developed using plant breeding techniques as describedabove. New inbred lines can be crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which of thosehave commercial potential.

The techniques of the present invention may be used to identifydesirable disease-resistant phenotypes by identifying genetic markersgenetically linked to an allele or locus conferring such a phenotype. Inaccordance with the invention, one of skill in the art may developmolecular marker assays based on, for example, SNPs and/or Indels in thespinach genome. In an embodiment, molecular marker assays useful toidentify DM-resistant spinach plants according to the invention may bedesigned based on a sequence scaffold from spinach chromosome 6. Suchtechniques may also involve phenotypic assays to identify desired plantseither alone or in combination with genetic assays, thereby alsoidentifying a marker genotype associated with the trait that may be usedfor production of new varieties with the methods described herein.

The invention provides for the production of cultivated spinach plantscomprising a combination of alleles conferring resistance to DM.Successful spinach production depends on attention to varioushorticultural practices. These include soil management with specialattention to proper fertilization, crop establishment with appropriatespacing, weed control, and the introduction of bees or other insects forpollination, irrigation, and pest management.

Spinach crops can be established from seed or from transplants.Transplanting can result in an earlier crop compared to a crop producedfrom direct seeding. Transplanting helps achieve complete plant standsrapidly, especially where higher seed costs, as with triploid seeds,make direct-seeding risky.

Development of Spinach Plants With Resistance to Downy Mildew

The present disclosure identifies alleles from cultivated spinach andcombinations thereof conferring broad-spectrum resistance to DM, as wellas sequence scaffolds from spinach chromosome 6 that can be used for thetracking and introgression of the loci into desirable germplasm, such asby marker-assisted selection and/or marker-assisted backcrossing.

The invention provides for the tracking and introduction of any suchalleles and/or any combination of such alleles with other resistanceloci into a given genetic background. One of ordinary skill willunderstand that resistance to DM conferred by alleles described hereinmay be introgressed from one genotype to another via marker-assistedselection. Accordingly, a germplasm source can be selected that hasresistance to DM. Using these alleles, a breeder may select a spinachplant with resistance to DM, or track such a phenotype during breedingusing marker-assisted selection for the region described herein.According to the invention, screens with flanking markers may besufficient to select progeny carrying desired DM resistance in pedigreesthat segregate for a single resistance allele. One of skill in the artwill also understand that markers can be complemented by phenotypicscreens, for example with differential races showing a compatibleinteraction with one allele and an incompatible interaction withanother, to select for individuals with desired resistance inpopulations segregating for two or more haplotypes.

Development of Spinach Hybrids With Resistance to Downy Mildew

As described herein, spinach plants in accordance with the presentinvention may comprise any heterozygous combinations of allele C, alleleA, and allele Vt to confer broad DM resistance. For example, in oneembodiment, alleles C and Vt can be present in a heterozygous spinachplant and confer resistance to Pfs races 1-8, 10-13, and to newlyoccurring Pfs isolate UA4712. In other embodiments, alleles A and Vt canbe present in a spinach plant and confer resistance to Pfs races 1-8 and10-14, or alleles A and C can be present together in a spinach plant andconfer resistance to Pfs races 1-8, 10-12, 14 and UA4712. Also inaccordance with the present invention is a three-way hybrid that isresistant to all described DM races and isolate UA4712. Such a spinachplant comprising one or two alleles as described herein may be crossedwith a second, distinct spinach plant comprising a second or thirdallele as described herein to produce a hybrid spinach plant comprisinga beneficial set of alleles as described herein.

The process of introgressing a novel resistance gene into acceptablecommercial types can be a difficult process and may be complicated byfactors such as linkage drag, epistasis, and low heritability. Theheritability of a trait is the proportion of the phenotypic variationattributed to the genetic variance, which varies between 0 and 1.0.Thus, a trait with heritability near 1.0 is not greatly affected by theenvironment. Those skilled in the art recognize the importance ofcreating commercial lines with horticultural traits having highheritability because these cultivars will allow growers to produce acrop with uniform market specifications.

One of ordinary skill will understand that the resistance allelesprovided herein can be combined for improved resistance, particularly infields where mixed populations of DM races may occur. According to theinvention, the alleles described herein may be introgressed into parentsof a hybrid via marker-assisted and/or phenotypic selection. Breedersmay select alleles that mutually complement race-specificity to achievebroad resistance to DM. The described resistance donors are crossed toinbreds with demonstrated combining ability. The F1 resulting from eachinitial cross is then backcrossed with the recipient genotype, i.e. withthe recurrent parent. According to the invention, individuals carryingthe resistance in the heterozygous phase are selected usingmarker-assisted and/or phenotypic selection. This backcross andselection step is repeated, for example, three times. Individualscarrying the desired resistance allele are then self-pollinated, forexample, for two generations through single seed descent. A breeder mayrecover inbred spinach plants carrying the desired resistance allelefrom the progeny of each pedigree, introduced into the same geneticbackground as respective recurrent parents, preferably at least 95%,96%, 97%, 98%, or 99% identical. Commercial, resistant cultivars aregenerated by crossing parental lines, each with a complementaryresistance allele, thereby creating hybrids with superior resistance.

According to the invention, multiple resistance donors may beindividually crossed to the same recipient genotype. Following thebackcross and selection steps described above, an inbred spinach plantcarrying a distinct resistance allele may be obtained that has beenintroduced into the same genetic background as the recurrent parent,preferably at least 95%, 96%, 97%, 98%, or 99% identical. One of skillin the art will understand that different resistance alleles may bemaintained in the same, fixed genetic background, i.e. in near-isogeniclines. The steps to create inbreds that are near-isogenic for resistancealleles can be applied to both inbred parents of any hybrid. Accordingto the invention, breeders may create three-way hybrids by crossing twonear-isogenic lines derived from one parental line to generate an F1seed parent. The F1 seed parent is crossed with the third parentcarrying the complementary resistance allele. The resulting three-wayhybrid is uniform, except for a complementary combination of DMresistance alleles. Hybrid plants consistently share the same geneticbackground as the recurrent parent, preferably at least 95%, 96%, 97%,98%, or 99% identical, and carry various combinations of resistancealleles. The alleles provided herein allow for the consistent productionof commercial varieties with broad DM resistance. Furthermore, thepresent invention provides alleles that can be used in differentcombinations to provide spinach plants for each area and/or region withdistinct DM populations. In addition, a single variety can be obtainedwith a mix of resistance alleles to reduce pathogen pressure whenresistance-breaking races or newly emerging isolates exist.

Genomic Region, Polymorphic Nucleic Acids, and Alleles Associated WithDM Resistance

Applicants have discovered three alleles from cultivated spinach, S.oleracea, that when present together in particular combinations in ahybrid (heterozygous) spinach plant, confer broad-spectrum resistance toDM. Using the methods outlined herein, these alleles were found to belocated on spinach chromosome 6 in a locus that may be defined bysequence scaffolds SF34732 and SF63815 on the distal position andSF59002, SF95487, and SF90906 on the proximal position, or sequences atleast 95% identical thereto, including sequences at least 96%, 97%, 98%,99%, or 100% identical thereto, as one of skill in the art wouldunderstand that polymorphisms may exist in such regions in differentpopulations. Polymorphisms that may be used to identify or diagnose thepresence of resistance alleles C, A, and/or Vt are shown in FIG. 3.Examples of such nucleotide sequences are provided in SEQ ID NOs:1-25.These sequences may be used in accordance with the invention to identifyor diagnose the presence or identity of a particular allele in a plantand thus identify plants that carry resistance to DM. One of skill inthe art will further appreciate that many genetic markers can be locatedthroughout the S. oleracea genome, and markers may be developed fromSNPs and/or Indels in flanking sequences of the sequences describedherein and in other fragments located throughout the S. oleracea genome.Such markers are useful in identifying the presence or absence of aresistance allele in accordance with the invention. Examples of markersin the spinach genome are described in, for example, Khattak et al.(Euphytica 148:311-318, 2006). The identification of alleles andDM-resistance conferring alleles as set forth herein, allows the use ofany other such markers in the same region and genetically linked (inlinkage disequilibrium) therewith.

The genomic region, alleles, and polymorphic markers identified hereincan be mapped relative to any publicly available physical or genetic mapto place the region described herein on such map. One of skill in theart would also understand that additional polymorphic nucleic acids asdescribed herein that are genetically linked to an allele associatedwith resistance to DM in spinach and that map within about 40 cM, 20 cM,10 cM, 5 cM, or 1 cM of an allele or a markers associated withresistance to DM in spinach may also be used.

The above markers and allelic states are therefore exemplary. One ofskill in the art would recognize how to identify spinach plants withother polymorphic nucleic acid markers and allelic states thereofrelated to resistance to DM in spinach consistent with the presentdisclosure. One of skill the art would also know how to identify theallelic state of other polymorphic nucleic acid markers located in thegenomic region(s) or linked to an allele or other markers identifiedherein, to determine their association with resistance to DM in spinach.

Genomic Regions, Polymorphic Nucleic Acids, and Genetic BackgroundAssociated with Recipient Parent.

Described herein are markers for use in identifying the presence orabsence of a resistance allele. In accordance with the invention,markers in the spinach genome that are not associated with or lacksignificant genetic linkage to the resistance locus allow the selectionof genetic background. One example of background selection is recoveryof the genome of a recipient parent in a recurring backcross scheme, anexample of which is provided herein. In recurrent selection, multiplerounds of mating between sibs carrying favorable characteristics arecarried out and selections made of progeny, allowing introduction of DMresistance-conferring alleles along with genetic diversity into apedigree while maintaining favorable attributes of one or more eliteparent. Examples of markers for spinach distributed genome-wide and thatmay be used in production of plants according to the methods of theinvention are described in, for example, Khattak et al. (Euphytica148:311-318, 2006). The identification of DM resistance-conferringalleles set forth herein, concurrent with background selection, allowsthe use of any marker in the same genetic interval and any marker inother genetic intervals of genomic fragments.

Introgression of a Genomic Locus Associated with Resistance to DM inSpinach

Provided herein are spinach plants (S. oleracea) comprising acombination of alleles that confer broad-spectrum resistance to DM andmethods of obtaining the same. In accordance with the invention,marker-assisted introgression involves the transfer of a chromosomalregion, defined by one or more markers, from one germplasm to a secondgermplasm. Offspring of a cross that contain an introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first germplasm (e.g.,germplasm with resistance to DM) and both linked and unlinked markerscharacteristic of the desired genetic background of a second germplasm.

Markers that are linked and/or either immediately adjacent to a locuscomprising an identified DM-resistance allele or locus as describedherein that permit introgression of an allele or locus in the absence ofextraneous linked DNA from the source germplasm containing the allele orlocus are provided herewith. Those of skill in the art will appreciatethat when seeking to introgress a smaller genomic region comprising anallele or locus associated with resistance to DM described herein, anyof the telomere proximal or centromere proximal markers that areimmediately adjacent to a larger genomic region comprising the allele orlocus can be used to introgress that smaller genomic region.

Spinach plants or germplasm comprising such an introgressed region thatis associated with resistance to DM wherein at least 10%, 25%, 50%, 75%,90%, or 99% of the remaining genomic sequences carry markerscharacteristic of plant or germplasm that otherwise or ordinarilycomprise a genomic region associated with another phenotype, aretherefore provided in specific embodiments. Furthermore, spinach plantscomprising an introgressed region where closely linked regions adjacentand/or immediately adjacent to a genomic region, allele or locus, and/ormarkers provided herewith that comprise genomic sequences carryingmarkers characteristic of spinach plants or germplasm that otherwise orordinarily comprise a genomic region associated with the phenotype arealso provided.

Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, Restriction Fragment LengthPolymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP),Simple Sequence Repeats (SSR), simple sequence length polymorphisms(SSLPs), Single Nucleotide Polymorphisms (SNP), Insertion/DeletionPolymorphisms (Indels), Variable Number Tandem Repeats (VNTR), RandomAmplified Polymorphic DNA (RAPD), isozymes, and others known to thoseskilled in the art. Marker discovery and development in crops providesthe initial framework for applications to marker-assisted breedingactivities (U.S. Patent Pub. Nos.: 2005/0204780; 2005/0216545;2005/0218305; and 2006/00504538). The resulting “genetic map” is therepresentation of the relative position of characterized loci(polymorphic nucleic acid markers or any other locus for which allelescan be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single-strand conformationalpolymorphism (Orita et al. Genomics, 8(2):271-278, 1989), denaturinggradient gel electrophoresis (Myers EPO 0273085, 1985), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg, Md.20877), although the widespread availability of DNA sequencing machinesoften makes it easier to just sequence amplified products directly. Oncethe polymorphic sequence difference is known, rapid assays can bedesigned for progeny testing, typically involving some version of PCRamplification of specific alleles (PASA, Sommer et al., Biotechniques12(1):82-87, 1992), or PCR amplification of multiple specific alleles(PAMSA, Dutton et al., Biotechniques 11(6):700-702, 1991).

As a set, polymorphic markers serve as a useful tool for fingerprintingplants to inform the degree of identity of lines or varieties (U.S. Pat.No. 6,207,367). These markers form the basis for determiningassociations with phenotypes and can be used to drive genetic gain. Incertain embodiments of methods of the invention, polymorphic nucleicacids can be used to detect in a spinach plant a genotype associatedwith resistance to DM, identify a spinach plant with a genotypeassociated with resistance to DM, and to select a spinach plant with agenotype associated with resistance to DM. In certain embodiments ofmethods of the invention, polymorphic nucleic acids can be used toproduce a spinach plant that comprises in its genome a combination ofalleles associated with resistance to DM. In certain embodiments of theinvention, polymorphic nucleic acids can be used to breed progenyspinach plants comprising a combination of alleles associated withresistance to DM.

Certain genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in a codominant fashionso that the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e., for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with a DM resistance phenotype.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (e.g., TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole-genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. Cold Spring Harbor Symp. Quant. Biol.51:263-273, 1986; European Patent No. 50,424; European Patent No.84,796; European Patent No. 258,017; European Patent No. 237,362;European Patent No. 201,184; U.S. Pat. No. 4,683,202; 4,582,788; and4,683,194), using primer pairs that are capable of hybridizing to theproximal sequences that define a polymorphism in its double-strandedform. Methods for typing DNA based on mass spectrometry can also beused. Such methods are disclosed in U.S. Pat. Nos. 6,613,509 and6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613; 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981; and 7,250,252 all of whichare incorporated herein by reference in their entireties. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include, but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944, where a sequence ofinterest is amplified and hybridized to probes, followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523, 2003; Cui et al.,Bioinformatics 21:3852-3858, 2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464, employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of the probes to the targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group that can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited to, those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is adjacent to a polymorphism to incorporatea detectable nucleotide residue upon extension of the primer. In certainembodiments, the SBE method uses three synthetic oligonucleotides. Twoof the oligonucleotides serve as PCR primers and are complementary tothe sequence of the locus of genomic DNA which flanks a regioncontaining the polymorphism to be assayed. Following amplification ofthe region of the genome containing the polymorphism, the PCR product ismixed with the third oligonucleotide (called an extension primer), whichis designed to hybridize to the amplified DNA adjacent to thepolymorphism in the presence of DNA polymerase and two differentiallylabeled dideoxynucleoside triphosphates. If the polymorphism is presenton the template, one of the labeled dideoxynucleoside triphosphates canbe added to the primer in a single base chain extension. The allelepresent is then inferred by determining which of the two differentiallabels was added to the extension primer. Homozygous samples will resultin only one of the two labeled bases being incorporated, and only one ofthe two labels will be detected. Heterozygous samples have both allelespresent and will direct incorporation of both labels (into differentmolecules of the extension primer), and thus both labels will bedetected.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787, in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR, forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism, while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle, a DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, an allele or locus of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays, as reviewed by R.F.Service Science 311:1544-1546, 2006 .

Markers used in accordance with the present invention should preferablybe diagnostic of origin in order for inferences to be made aboutsubsequent populations. Experience to date suggests that SNP markers maybe ideal for mapping because the likelihood that a particular SNP alleleis derived from independent origins in the extant populations of aparticular species is very low. As such, SNP markers appear to be usefulfor tracking and assisting introgression of alleles or loci.

Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which spinach plants canbe regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, “DM” or “downy mildew” refers to a disease of plants,such as spinach, caused by a pathogen from the genus Peronospora, inparticular Peronospora farinosa f. sp. Spinaciae (Pfs).

As used herein, “race” refers to an officially designated strain ofPeronospora farinosa f. sp. spinaciae (Pfs) that can cause DM. As usedherein, “isolate” refers to a newly occurring strain of Peronosporafarinosa f. sp. spinaciae (Pfs) that can cause DM, and has not yet beenofficially named. A spinach plant with resistance to DM according to thepresent invention carries a combination of alleles selected from A, C,and Vt. The DM resistance may be to one or more known races ofPeronospora farinosa f. sp. spinaciae, or may be to one or more isolatesof Peronospora farinosa f. sp. spinaciae. In another embodiment, a plantof the invention may be defined as resistant to at least Peronosporafarinosa f. sp. spinaciae Pfs 7, 8, 10, 11, 12, 13, and/or 14.

As used herein, the terms “pedigree,” “population,” and “progeny” mean acollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “resistance” means evasion and/or reduction ofpathogen infection by plant innate immunity, which can be shown by theabsence and/or reduction of disease symptoms when compared to asusceptible plant.

As used herein, the term “susceptible” means the infection of a plant bya pathogen, resulting in disease symptoms.

As used herein, the term “compatible interaction” means the infection ofa susceptible plant by a pathogen, resulting in disease symptoms.

As used herein, the term “incompatible interaction” means the evasionand/or reduction of infection of a resistant plant by a pathogen, whichcan be shown by the absence and/or a reduction of disease symptoms.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, the term “heterozygous phase” means a diploid plant,such as spinach, that carries two distinct copies of an allele.

As used herein, the term “homozygous phase” means a diploid plant, suchas spinach, that carries two identical copies of an allele.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can therefore be achieved through plant breeding methodsand/or by molecular genetic methods. Such molecular genetic methodsinclude, but are not limited to, various plant transformation techniquesand/or methods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, the term “denoting” when used in reference to a plantgenotype refers to any method whereby a plant is indicated to have acertain genotype. This includes any means of identification of a planthaving a certain genotype. Indication of a certain genotype may include,but is not limited to, any entry into any type of written or electronicmedium or database whereby the plant's genotype is provided. Indicationsof a certain genotype may also include, but are not limited to, anymethod where a plant is physically marked or tagged. Illustrativeexamples of physical marking or tags useful in the invention include,but are not limited to, a barcode, a radio-frequency identification(RFID), a label, or the like.

Deposit Information

A deposit was made of at least 2500 seeds of Spinacia oleraceaaccessions designated SMBS011-1162M, SMB-66-1143M, and SSB-66-1131M. Thedeposits were made with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209 USA. The depositswere assigned ATCC Accession Nos. PTA-120472, PTA-12486, and PTA-12041,respectively. The dates of deposit of these accessions were Jul. 17,2013, Feb. 2, 2012, and Aug. 19, 2011, respectively. Access to thedeposits will be available during the pendency of the application topersons entitled thereto upon request. The deposits will be maintainedin the ATCC Depository, which is a public depository, for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced ifnonviable during that period. Applicant does not waive any infringementof their rights granted under this patent or any other form of varietyprotection, including the Plant Variety Protection Act (7 U.S.C. 2321 etseq.).

EXAMPLES

The following disclosed embodiments are merely representative of theinvention which may be embodied in various forms. Thus, specificstructural, functional, and procedural details disclosed in thefollowing examples are not to be interpreted as limiting.

Example 1 Identification of Novel Resistance to DM Races in Spinaciaoleracea

A screen was performed on S. oleracea germplasm using the disease assaydescribed in Example 5. S. oleracea accessions in an internal genebankwere screened. In addition, publicly available spinach hybrids that wereeither fully susceptible, or carried a combination of resistancespecificities were included in the trial as susceptible and resistancechecks. Infection was performed with Peronospora farinosa f. sp.spinaciae (Pfs) races 7, 8, and 10-14 to complement historic data onraces 1-6. Race 9 is no longer present in the field. In addition tostrains with a race designation, a resistance-breaking isolate,collected in California (US) in the spring of 2013 was included. Thisisolate is currently referred to as UA4712 by the International WorkingGroup on Peronospora and is a probable candidate for race Pfs 15.

The response of control entries to infection is presented in Table 1 andis consistent with data published elsewhere (Correll et al., Eur J PlantPathol 129:193-205, 2011). Despite the contribution of resistancespecificities by both parents, all commercial hybrid checks included inthe trial failed to respond with resistance to all isolates. Forexample, variety Lion is susceptible to race 10, Lazio is susceptible toraces 11-14, and Pigeon is susceptible to race 14. Resistance to bothraces 10 and 14 was not observed in any single hybrid, and a combinationof resistance to races 13 and 14 was only observed for the hybrid Lion.

Among the S. oleracea accessions in the internal genebank, three lineswere found to have a complementary combination of resistances. Inbredline SMBS011-1162M, carrying an allele identified as allele A, wasresistant to multiple races, including races 13 and 14. A second inbredline, SSB-66-1131M, included an allele designated Vt, was found to beresistant to races 1-13. A final inbred line, SMB-66-1143M, whichincludes an allele identified as allele C, was shown to be resistant tonew isolate UA4712, in addition to races 1-10. Each of the identifiedalleles were found to confer DM resistance to different combinations ofraces.

TABLE 1 Phenotypic response of control and experimental entries toinfection by Pfs races 1-14 and isolate UA4712. LINES A Vt C AccessionCOMMERCIAL HYBRIDS (SMBS011- (SSB-66- (SMB-66- Race Viroflay ResistoflayClermont Campania Boeing Califlay Whale Lion Lazio Pigeon 1162M) 1131M)1143M) Pfs 1 S R R R R R R R R R R R R Pfs 2 S R R R R S S R R R S R RPfs 3 S S R R R R R R R R R R R Pfs 4 S S R R R S S R R R S R R Pfs 5 SS S R R R R R R R R R R Pfs 6 S S S S R S S R R R S R R Pfs 7 S S S R RS S R R R R R R Pfs 8 S S S S S R R R R R R R R Pfs 10 S S S S S S S S RR S R R Pfs 11 S S S R R R R R S R R R S Pfs 12 S S S S S R R R S R R RS Pfs 13 S S S S R S S R S R R R S Pfs 14 S S S S S R R R S S R S SUA4712 S S R S, susceptible response, with an average percentage ofsporulating leaf surface estimated above 85%; R, resistance,demonstrated by no or little (<3%) sporulation.

Example 2 Mapping Resistance to DM

Irish et al. (2008) identified a co-dominant marker, designated Dm-1,which was closely linked to the resistance locus RPF1 from S. oleracea.The genetic distance between Dm-1 and RPF1 was estimated to be 1.7 cM.Further efforts to fine-map the Dm-1 marker relative to the RPF1 locusgenerated sequence resources in the vicinity of the Dm-1 marker, but didnot result in cloning RPF1 (Yang et al., Initial fine-mapping of thespinach downy mildew resistance locus RPF1, University of Arkansas,2013, 102 pages; 1536814). Given the absence of a causal polymorphismand the distance between Dm-1 and RPF1, association may be lost duringbackcrossing or recurrent selection. Therefore, markers flankingresistance on both sides were needed for tracking resistance alleles andfurther fine-mapping.

Spinach lines carrying one of the three resistance alleles, A, Vt, or C,were individually crossed to a line that exclusively harbored resistanceto races Pfs 1-4. Resistance to most races is inherited in a dominantfashion, based on the absence of symptoms upon infection of F1individuals with races that differentiate between resistance from bothparents. In contrast, resistance from allele A to races 7 and 13 appearsto be recessive. F3 families were obtained through single seed descentfrom the separate bi-parental F1 crosses and screened phenotypically.The disease assay included 74 F3 families segregating for allele A,which were infected with Pfs race 8; 107 F3 families segregating forallele Vt challenged with races 7, 10, and 12; and 40 F3 familiessegregating for allele C, which were inoculated with race 10.Interestingly, when Vt-derived families demonstrated resistance to onerace, immunity to the other two races was also observed.

Based on public information, the likely position of sequence scaffoldsdesignated SF34732, SF59002, SF62749, SF63815, SF90906, SF95487, andSF178637 co-located with DM resistance. Examples of nucleotide sequencescorresponding to alleles C, A, and Vt conferring resistance to DM areprovided as SEQ ID NOs:1-25. Additionally, polymorphisms betweenindividual alleles of scaffolds are shown in FIG. 3. Single nucleotidepolymorphisms (SNPs) derived from these scaffolds were selected based onbeing polymorphic in one or more mapping populations. The SNPs wereconverted to TaqMan™ assays and used for genotyping F2 individuals ofthe respective F3 families of each population. Linkage disequilibriumwas tested, and the genetic distances among SNPs and phenotypes wereestimated using Joinmap (Stam, Plant J 3:739-744, 1993). Associationanalysis confirmed linkage of sequence scaffolds and resistance byassembly of single groups. This result was found to be independent ofthe source or race used for mapping (FIG. 1). Observed recombinationfrequencies and derived genetic distances varied due to the distinctsample size of each population. Scaffolds SF59002 and SF95487 appearedto co-segregate in every population. The order of scaffolds and geneticmarkers was consistent for populations segregating for allele Vt orallele C. However, the exact position of SF62749 relative to, forexample, scaffolds SF59002 and SF90906 remains ambiguous. Finally,converted and mapped SSR markers, which were published by Khattak et al.(Euphytica 148:311-318, 2006), identified the linkage group as publicLG6.

Example 3 Quantitative Effects of DM Resistance Alleles

The genotype and phenotypes collected in Example 2 were used in aquantitative analysis with mapQTL, applying standard interval mappingsettings (Van Ooijen, 1996). A major locus for resistance to Peronosporafarinosa f. sp. spinaciae race 8 was detected in the mapping populationsegregating for allele A. Evidenced by a LOD peak of 57.51, the traitwas highly associated with markers derived from scaffolds SF59002,SF95487, and SF63815 (Table 2). The locus appeared to have a majoreffect, explaining 97.2% of the observed variance.

TABLE 2 Quantitative analysis of resistance allele A to Pfs race 8. maplod iter mu_A mu_H mu_B var % expl add dom locus — — — — — — — — — — — 040.53 4 94.4444 50 2.63158 92.0657 92 45.9064 1.46199 SF90906a 0.7 46.424 97.0588 50 2.63158 63.8022 94.4 47.2136 0.154799 SF90906b 1.4 57.51 4100 50 2.63158 32.0057 97.2 48.6842 −1.31579 SF59002 1.4 57.51 4 100 502.63158 32.0057 97.2 48.6842 −1.31579 SF95487 1.4 57.51 4 100 50 2.6315832.0057 97.2 48.6842 −1.31579 SF63815 5.7 31.74 4 94.1176 50 2.77778159.093 86.1 45.6699 1.55229 SF62749

A second analysis with source Vt (SSB-66-1131M) for resistance to Pfsraces 7, 10, and 12 also identified a resistance locus (Tables 3, 4, and5). Independent of the isolate used, the locus appeared to be stronglylinked to scaffolds SF95487 and SF59002, evident by a LOD peak of 91.81.This locus had a major effect, explaining nearly all phenotypicvariance. Strikingly, the loci identified in resistant sources A(SMBS011-1162M) and Vt (SSB-66-1131M) were collinear.

TABLE 3 Quantitative analysis of resistance allele Vt to Pfs race 7. maplod iter mu_A mu_H mu_B var % expl add dom locus — — — — — — — — — — — 024.43 4 88 52.7273 9.25926 408.188 65.1 39.3704 4.09764 SF34732a 0.625.6 4 88.4615 51.8519 9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.791.81 4 98.0769 50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 498.0769 50 0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.444450.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 489.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749

TABLE 4 Quantitative analysis of resistance allele Vt to Pfs race 10.map lod iter mu_A mu_H mu_B var % expl add dom locus — — — — — — — — — —— 0 24.43 4 88 52.7273 9.25926 408.188 65.1 39.3704 4.09764 SF34732a 0.625.6 4 88.4615 51.8519 9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.791.81 4 98.0769 50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 498.0769 50 0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.444450.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 489.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749

TABLE 5 Quantitative analysis of resistance allele Vt to Pfs race 12.map lod iter mu_A mu_H mu_B var % expl add dom locus — — — — — — — — — —— 0 24.43 4 88 52.7273 9.25926 408.188 65.1 39.3704 4.09764 SF34732a 0.625.6 4 88.4615 51.8519 9.25926 388.21 66.8 39.6011 2.99145 SF34732b 11.791.81 4 98.0769 50 0 22.4659 98.1 49.0385 0.961538 SF95487 11.7 91.81 498.0769 50 0 22.4659 98.1 49.0385 0.961538 SF59002 13.8 55.39 4 94.444450.9259 1.92308 107.703 90.8 46.2607 2.74217 SF90906 19.8 31.02 489.6552 51.0638 11.2903 307.4 73.7 39.1824 0.591082 SF62749

A study for resistance from allele C was performed with Pfs race 10.Similar to previous results, a major locus for resistance appeared to besignificantly associated with scaffolds SF34732 and SF6381 (Table 6).

TABLE 6 Quantitative analysis of resistance allele C to Pfs race 10. maplod iter mu_A mu_H mu_B var % expl add dom locus — — — — — — — — — — — 0perfect fit 3 100 50 0 0 100 50 0 SF34732 0 perfect fit 3 100 50 0 0 10050 0 SF63815 1.3 28.02 4 100 46.6667 0 58.3333 96 50 −3.33333 SF954871.3 28.02 4 100 46.6667 0 58.3333 96 50 −3.33333 SF59002 2.6 22.3 4 10046.4286 6.25 112.723 92.3 46.875 −6.69643 SF90906 8.4 12.19 4 10047.2222 8.33333 361.111 75.4 45.8333 −6.94444 SF62749 22.1 6.14 496.6667 47.2222 28.5714 724.504 50.7 34.0476 −15.3968 SF178637

DM resistance mapped to Chromosome 6 independent of the source orisolate used. The resistance loci were delineated by common markers, onthe distal position typically sequence scaffolds SF34732 and SF63815 andon the proximal position typically sequence scaffolds SF59002, SF95487and SF90906.

Example 4 Deployment of Resistance Alleles A, Vt, and C in Hybrids

The resistance alleles A, C and Vt were introduced into a susceptiblehybrid. The inbred SMBS011-1162M was used as a donors for allele A, theline SSB-66-1131M for Vt, and for allele C, SMB-66-1143M was used as asource. Each of the alleles was introgressed separately into both inbredparents of the susceptible hybrid, following breeding methods known inthe art. Next, a first spinach plant which is homozygous for allele A(A/A) was crossed to a second spinach plant (C/C) to generate a hybrid F1 which was heterozygous for alleles A and C (FIG. 2A). This hybrid wasreferred to as Single Hybrid A, or as F1 (A/C). F 1(A/C) was screenedwith known races and isolate UA4712 of Peronospora farinosa f. sp.spinaciae, as described in Example 5. The hybrid was found to beresistant to all Pfs races, with the exception of race 13 (Table 7).Hybrid A is also resistant to the newly emerging isolate UA4712.

Hybrid B carrying alleles C and Vt (FIG. 2B) was screened as describedand found to be resistant to all Pfs races except Pfs 14 (Table 7). Thisis important, as Pfs race 14 does not occur in Europe. A combination ofalleles C and Vt in a spinach plant provides a fully resistant varietyfor the European market.

Hybrid C with alleles A and Vt was resistant to all Pfs races exceptisolate UA4712 (Table 7). This is also significant, since thedistribution of a new isolate is initially limited to few locations.F1(A/Vt) is resistant to all currently named Pfs races.

In addition to biparental hybrids, three-way hybrids were generated.Three-way hybrids are produced by crossing an F1 seed parent to aninbred parent P2. The F1 seed parent results from a cross between twonear-isogenic lines of P1, each carrying a distinct resistance allele.The inbred parent P2 carries a third, complementary allele. A three-wayhybrid allows the deployment of all three identified alleles in a mixedpopulation of diploid hybrid plants. Three versions of the 3-way hybridare possible (FIG. 2B). Each cross results in a population of hybridplants with resistance to all known Pfs races and isolate UA4712.However, the frequency of each allele will depend on how the three-waycross is structured.

Novel resistance originating from the genetic variety contained withincultivated spinach, Spinacia oleracea was identified. This resistance issurprising because genetic diversity in cultivated crops is typicallynarrow (Fernie et al., Curr. Opinion Pl. Biol. 9:196-202, 2006). Theidentification of the three unique resistance alleles, A, Vt, and/or C,in the three spinach lines provides for race-specific DM resistancebreeding, with only intra-specific S. oleracea crosses. Additionally,these alleles allow stacking of resistance in single hybrid combinationsthat are unsurpassed in combining novel attributes, including resistanceto all known Pfs races, to the new isolate UA4712, or resistance to alldescribed European DM populations. Markers associated with the threeresistance alleles allow introduction of the described and otherDM-resistance alleles in any cultivated spinach hybrid. Finally, athree-way hybrid method is disclosed to allow for the development ofadditional DM resistant varieties.

TABLE 7 Phenotypic response of testcrosses to infection of leaf discs byPfs races 1-8, 10-14 and isolate UA4712 TEST CROSS Pfs 1 Pfs 2 Pfs 3 Pfs4 Pfs 5 Pfs 6 Pfs 7 Pfs 8 Pfs 10 Pfs 11 Pfs 12 Pfs 13 Pfs 14 UA4712 F1(A/C) R R R R R R R R R R R S R R F1 (C/Vt) R R R R R R R R R R R R S RF1(AxVt) R R R R R R R R R R R R R S “S” indicates a susceptibleresponse. “R” indicates resistance

Example 5 Assays for Screening Spinach Accessions for Resistance to DM

A test was utilized for screening spinach accessions for resistance todowny mildew (DM) that originated from the International Union for theProtection of New Varieties of Plants (UPOV). The “Protocol for Tests onDistinctness, Uniformity, and Stability of Spinacia oleracea L.Spinach,” UPOV Code: SPINA_OLE, CPVO-TP/055/5 was adopted and into forceon Feb. 27, 2013. The protocol is as follows:

Races of Peronospora farinosa f. sp. spinaciae are maintained on livinghost plants, obtainable from Naktuinbouw (P.O. Box 40, NL-2370 AA,Roelofarendsveen, Netherlands, naktuinbouw.com), or plant material withspores stored at −20° C. for a maximum of one year.

Execution of test: Growth stage of plants: First cotyledons/leaf,eleven-day-old plants; Temperature: 15° C. during day/12° C. duringnight; Light: 15 hours per day, after emergence; Growing method: In soilin pots or trays in a glasshouse or growth chamber.

Method of inoculation: Sporulating leaves, taken from host plants thatwere infected seven days before, are thoroughly rinsed with sterile tapwater (maximum 150 ml water per 224 plants). The spore suspension isfiltered through cheesecloth and sprayed on test plants until theinoculum covers the leaves but does not run off. 150 ml of suspension isenough for up to 3×224 plants. Spore density should be 20,000 to 100,000conidia/ml water. The spore suspension should be used fresh. As spinachdowny mildew is wind-borne, sporulating plants should be kept in closedcontainers or isolated chambers to prevent any cross-contamination.

Resistant controls are needed in each multiplication and in each test toensure the race identity. Light and humidity conditions during seedlingdevelopment and incubation are critical. Optimal humidity ofapproximately 80-90% RH allows plant growth and fungal growth; stronglight inhibits spore germination and infection. The test should becarried out in wintertime with protection against direct sunshine. Afterinoculation, the plants should remain under plastic for three days.After this time, the plastic should be slightly raised during thedaytime.

Duration of test: Multiplication harvest spores 7 days afterinoculation; Sowing to inoculation: 11 days; Inoculation to reading: 10days; Number of plants tested: 56 plants; Evaluation of infection:Resistance is usually complete; sometimes necrotic spots are visible asa result of infection. Susceptible plants show varying degrees ofsporulation. Sporulation is visible as a grey covering on leaves,starting on the more humid abaxial side.

Differential varieties to identify races: Races Pfs: 1-8 and 10-13 ofPeronospora farinosa f. sp. spinaciae are defined with a standard set of“differential varieties” according to Table 8.

TABLE 8 Differential varieties to identify races: Races Pfs: 1-8 and10-13 of Peronospora farinosa f. sp. spinaciae. Differential varietyPfs: 1 Pfs: 2 Pfs: 3 Pfs: 4 Pfs: 5 Pfs: 6 Pfs: 7 Pfs: 8 Pfs: 10 Pfs: 11Pfs: 12 Pfs: 13 Viroflay S S S S S S S S S S S S Resistoflay R R S S S SS S S S S S Califlay R S R S R S S R S R R S Clermont R R R R S S S S SS S S Campania R R R R R S R S S R S S Boeing R R R R R R R S S R S RLion R R R R R R R R S R R R Lazio R R R R R R R R R S S S R, resistancepresent; S, resistance absent (susceptible)

1. A Spinacia oleracea spinach plant of a cultivated variety comprisingin its genome allele A, wherein said plant comprises a DNA sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, and SEQID NO:22, and comprises the DNA sequence of SEQ ID NO:5 or SEQ ID NO:10,and wherein a sample of seed comprising said allele A has been depositedunder ATCC Accession No. PTA-120472.
 2. A Spinacia oleracea spinachplant of a cultivated variety comprising in its genome a heterozygouscombination of alleles that confers broad-spectrum resistance toPeronospora farinosa f. sp. Spinaciae (Pfs), wherein the combination ofalleles comprises two alleles selected from the group consisting ofallele A, allele Vt, and allele C, and wherein a plant comprising alleleA comprises a DNA sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:19, and SEQ ID NO:22, and comprises the DNA sequence ofSEQ ID NO:5 or SEQ ID NO:10, wherein a plant comprising allele Vtcomprises a DNA sequence selected from the group consisting of SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 andcomprises the DNA sequence of SEQ ID NO: 20 or SEQ ID NO: 23, andwherein a plant comprising allele C comprises a DNA sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:18, and SEQ ID NO:21and comprises the DNA sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ IDNO:12, and SEQ ID NO: 15, and wherein a sample of seed comprising saidallele A, allele Vt, and allele C has been deposited under ATCCAccession No. PTA-120472, ATCC Accession No. PTA-12041, and ATCCAccession No. PTA-12486, respectively.
 3. The spinach plant of claim 2,wherein the broad-spectrum resistance comprises resistance to at least10 races of Peronospora farinosa f. sp. spinaciae (Pfs).
 4. (canceled)5. The spinach plant of claim 2, wherein the combination of allelescomprises: (a) alleles A and C; (b) alleles A and Vt; or (c) alleles Cand Vt.
 6. The spinach plant of claim 3, wherein the plant is resistantto: (a) Peronospora farinosa f. sp. Spinaciae races, 7, 8, 10, 11, 12,14, and isolate UA4712; (b) Peronospora farinosa f. sp. Spinaciae races,7, 8, 10, 11, 12, 13, and 14; or (c) Peronospora farinosa f. sp.Spinaciae races, 7, 8, 10, 11, 12, 13, and isolate UA4712. 7.-8.(canceled)
 9. The spinach plant of claim 1, wherein said plant is aninbred.
 10. The spinach plant of claim 1, wherein said plant is ahybrid.
 11. The spinach plant of claim 1, comprising recessiveresistance to Peronospora farinosa f. sp. Spinaciae races 7 and
 13. 12.A seed that produces the plant of claim
 2. 13. A plant part of the plantof claim 2, wherein the plant is a leaf, stem, root, flower or cell. 14.A method of producing a seed of a spinach plant with broad spectrumresistance to Peronospora farinosa f. sp. Spinaciae comprising: (a)crossing a first spinach plant and a second spinach plant, wherein thefirst and second spinach plants collectively comprise at least twoalleles selected from the group consisting of allele A, allele Vt, andallele C, wherein a plant comprising allele A comprises a DNA sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:19, and SEQID NO:22, and comprises the DNA sequence of SEQ ID NO:5 or SEQ ID NO:10,wherein a plant comprising allele Vt comprises a DNA sequence selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQID NO:14, and SEQ ID NO:17 and comprises the DNA sequence of SEQ ID NO:20 or SEQ ID NO: 23, and wherein a plant comprising allele C comprises aDNA sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:18, and SEQ ID NO:21 and comprises the DNA sequence of SEQ ID NO:4,SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO: 15, and wherein a sample ofseed comprising said allele A, allele Vt, and allele C has beendeposited under ATCC Accession No. PTA-120472, ATCC Accession No.PTA-12041, and ATCC Accession No. PTA-12486, respectively; (b) obtainingat least a first seed resulting from said crossing that comprises aheterozygous combination of said alleles that confers broad-spectrumresistance to Peronospora farinosa f. sp. Spinaciae.
 15. The method ofclaim 14, wherein the method further comprises obtaining a population ofseed resulting from said crossing that comprises a heterozygouscombination of said alleles that confers broad-spectrum resistance toPeronospora farinosa f. sp. Spinaciae.
 16. The method of claim 14,wherein: (a) the first spinach plant comprises allele A and the secondspinach plant comprises allele C; (b) the first spinach plant comprisesallele A and the second spinach plant comprises allele Vt; (c) the firstspinach plant comprises allele C and the second spinach plant comprisesallele Vt; (d) the first spinach plant or the second spinach plant ishomozygous for said allele A, allele Vt, or allele C; or (e) the firstspinach plant and the second spinach plant are homozygous for said twoalleles selected from the group consisting of allele A, allele Vt, andallele C.
 17. A seed produced by the method of claim
 14. 18. The seed ofclaim 17, wherein: (a) the first allele is allele A and the secondallele is allele C; (b) the first allele is allele A and the secondallele is allele Vt; or (c) the first allele is allele C and the secondallele is allele Vt.
 19. A DNA sequence selected from the groupconsisting of SEQ ID NOs:1-25.
 20. A method of introducing resistance toPeronospora farinosa f. sp. Spinaciae into a spinach plant comprising:(a) crossing a first spinach plant comprising in its genome a firstallele selected from the group consisting of A, Vt, and C, with a secondspinach plant, wherein a plant comprising allele A comprises a DNAsequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:19, and SEQ ID NO:22, and comprises the DNA sequence of SEQ ID NO:5or SEQ ID NO:10, wherein a plant comprising allele Vt comprises a DNAsequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 and comprises the DNAsequence of SEQ ID NO: 20 or SEQ ID NO: 23, and wherein a plantcomprising allele C comprises a DNA sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:18, and SEQ ID NO:21 and comprisesthe DNA sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:12, and SEQ IDNO: 15, and wherein a sample of seed comprising said allele A, alleleVt, and allele C has been deposited under ATCC Accession No. PTA-120472,ATCC Accession No. PTA-12041, and ATCC Accession No. PTA-12486,respectively; (b) selecting at least one progeny plant that comprisessaid first allele for resistance to Peronospora farinosa f. sp.Spinaciae, by detecting the presence in the genome of said progeny plantthe DNA sequence of claim
 19. 21. The method of claim 20, wherein saidcrossing comprises producing a population of progeny plants.
 22. Themethod of claim 20, comprising screening the progeny plants for thepresence of at least two nucleic acid sequences selected from the groupconsisting of SEQ ID NOs: 1-25.
 23. A plant produced by the method ofclaim
 20. 24. A method of identifying a plant comprising a desiredallele conferring resistance to Peronospora farinosa f. sp. Spinaciae,the method comprising detecting in the genome of said plant the DNAsequence of claim
 19. 25. The method of claim 24, further defined ascomprising detecting in the genome of said plant at least twopolymorphic nucleic acid sequences selected from the group consisting ofSEQ ID NOs:1-25, wherein the presence of the polymorphic nucleic acidsequences are indicative of the presence in the plant of at least twoalleles conferring resistance to Peronospora farinosa f. sp. Spinaciaeselected from of alleles A, Vt, and C, wherein a plant comprising alleleA comprises a DNA sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:19, and SEQ ID NO:22, and comprises the DNA sequence ofSEQ ID NO:5 or SEQ ID NO:10, wherein a plant comprising allele Vtcomprises a DNA sequence selected from the group consisting of SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17 andcomprises the DNA sequence of SEQ ID NO: 20 or SEQ ID NO: 23, andwherein a plant comprising allele C comprises a DNA sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:18, and SEQ ID NO:21and comprises the DNA sequence of SEQ ID NO:4, SEQ ID NO:9, SEQ IDNO:12, and SEQ ID NO: 15, and wherein a sample of seed comprising saidallele A, allele Vt, and allele C has been deposited under ATCCAccession No. PTA-120472, ATCC Accession No. PTA-12041, and ATCCAccession No. PTA-12486, respectively.
 26. A plant obtained by themethod of claim
 24. 27. A spinach plant, cell or cell containing theplant part of claim 2.